Device and system for transfer of material

ABSTRACT

The invention concerns a device for collection of magnetic particles. The invention further concerns a system for transfer of material from a plurality of source vessels to a plurality of target vessels, the device comprising a plurality of collecting members each of which can be manipulated independently. The invention further concerns a method for detection of biological entities being fluorescent label, wherein the entities are bound to magnetic particles. The fluorescent emission is concentrated by clustering the magnetic particles utilizing magnetic force.

CROSS REFERENCE TO RELATED APPLICATION

The present application is the national stage under 35 U.S.C. 371 ofPCT/IL99/00078, filed Feb. 5, 1999.

FIELD OF THE INVENTION

The present invention concerns a device and system for the transferand/or manipulation of liquids or particles. The present inventionfurther concerns a method of detection of biological entities usingmagnetic particles.

BACKGROUND OF THE INVENTION

In recent years, with the advance of automatization in laboratorytechniques, many assays, reactions, diagnostic procedures and synthesistechniques, are carried out by the transfer of a plurality of liquidsamples, simultaneously, from one array of liquid-containing wells toanother. Typically, these are arrays of 5, 8, 16, 25, 96, 384, or 1536liquid-containing wells. In order to transfer, add, collect or mixliquids, or particles to present in all the wells in the arraysimultaneously, various multi-collector systems have been devised. Themost commonly used is a multi-pipetor which collects liquid from anarray of source wells to an array of target wells, simultaneously, byapplication or release of application, respectively, of vacuum force.However, in all known multi-pipetor devices, each individual pipetorcapable of collecting or releasing liquid from the well is connected byvacuum force to all other pipetors so that all samples in the well arecollected and released at once (Valerio et al., Analytical Biochemistry,197:168-177 (1991)).

Magnetic particles are used for a variety of separation, purification,and isolation techniques in connection with chemical or biologicalmolecules. In those techniques, a magnetic particle is coupled to amolecule capable of forming a specific binding (hereinafter “affinitybinding”) with a molecule in a biological sample, which is to beisolated, purified or separated. The biological sample is then broughtinto contact with the magnetic particle and those biological moleculeswhich bind to the magnetic particles are then isolated by application ofa magnetic field.

Such magnetic separation techniques have been employed to sort cells, torecover antibodies or enzymes from a solution, to purify proteins usingaffinity techniques, and to remove unwanted particles from suspension,for example, to remove cancer cells ex vivo, from a cell preparationwhich is then injected into a patient (Pourfarzaneh, M. et al., “The useof magnetizable particles in solid phase immunoassay in methods ofbiochemical analysis” 28:267-295 (1982)).

For the purpose of using magnetic particles, various devices have beendeveloped in order to transfer the magnetic particles from one locationto another, for example from one reaction vessel to another reactionvessel.

U.S. Pat. No. 4,292,920 discloses a device for transferring, bybio-magnetic attraction, antigen-antibody adsorbent material from onereaction mixture to the other. The device may comprise a single ormultipin arrangement, corresponding to a well arrangement, which iscapable of attracting by magnetic force magnetic particles. By oneembodiment, the pin is connected to an electromagnet, and by turning theelectromagnet on and off the pin becomes magnetized, or non-magnetized,respectively.

U.S. Pat. No. 5,567,326 discloses an apparatus and method for separatingmagnetically responsive particles from a nonmagnetic test medium inwhich they are suspended. The device comprises a plurality ofnonmagnetic pins (termed “magnetic field directing elements”) arrangedin an array, and a magnet positioned normal to said array. Placing themagnet on the array of pins, renders all the pins in the array magneticcausing particles to be attracted to them, and thus collecting them; andremoving the magnet causes the pins to become non-magnetic, andconsequently the magnetic particles are released from the pins.

The drawbacks of the above devices and apparatuses reside in the factthat the magnetic pins come into direct contact with the magneticparticles, so that if rinsing and sterilization is required, the wholeapparatus or device has to be washed which procedure is expensive andtime consuming. Furthermore, the collection of particles is notefficient since in such a construction, due to surface tension forces,some of the particles remain in the suspension.

Another drawback of the prior art devices reside in the fact that wherea multi pin device is used to collect magnetic particles from aplurality of wells, the particles from all the wells have to becollected at once in an “all or none” fashion, and it is not possible toselectively collect particles only from some wells in an array.

Magnetic particles were also used for detection purposes, for examplefor DNA purification for detection purposes, using a method similar toreverse hybridization blot system. However here the specificoligonucleotide probe was attached to a paramagnetic particle instead ofa sheet membrane. The target DNA, which contains the complementarysequence of the probe, hybridizes to the probe that is attached to thebead and is then magnetically removed from the solution, washed andcollected (Fry et al., Bio Techniques, 13(1) 124-131 (1992)). Couplingof the polynucleotides to the magnetic particles can be carried out forexample according to the teaching of Day et al. (Biochem. J. 278,735-740 (1991)). Immobilization of nucleic acid sequences on magneticbeads can also be carried out utilizing the streptavidin—biotentechnology (Uhlen M., Nature, 340, 773-739 (1989)).

GLOSSARY

The following terms will be used at times throughout the specification:

“Material”—the contents of a vessel which is tranferred from a firstplurality of vessels termed “source vessels” (see below) to a secondplurality of vessels termed “target vessels” (see below). Typically, a“material” is a small amount of liquid, solid particles (such as beads),for example contained in a liquid or magnetic particles.

“Plurality of vessels”—two or more material-holding vessels. Theplurality can be present in one “array of vessels” (see below), forexample each plurality may be a line or column in a single array.Alternatively, the plurality may be the array of vessels itself forexample a 96-well array.

“Array of vessels”—a plurality of voids present in a single constructwhich holds the material. Typically the array of vessels is an array ofwells. State of the art wells have an 8, 16, 25, 96, 384 or 1536—wellarrangement.

“Source vessels”—a plurality of vessels from which material iscollected.

“Target vessels”—a plurality of vessels to which material is released.

“Transfer”—an action of withdrawing and holding (i.e. collecting) thematerial from one plurality of vessels (source) and releasing thematerial to another plurality of vessels (target).

“Collecting member”—a component of the system of the invention capableof collecting (upon activation) and releasing (upon deactivation)material from a single vessel for example from a single well. The systemcomprises a plurality of individual collecting members each capable ofbeing activated and deactivated independently.

“Activated state” (“activated”) “deactivated state”“Deactivate”—a changeof property of the individual collecting member which can cause it tocollect or release material, respectively, for example, by applicationand removal of vacuum force. Another example is creation of an activatedstate by placing a magnetic-field providing member in a fist positionwhere it is present at the distal-collecting end of the device anddeactivated state is created by moving said member to a second positionwhere it is spaced from said distal-collecting end. In the firstposition magnetic particles (the material) one collected and in thesecond position magnetic particles are relevant. Another option is acollecting member comprising an electromagnet when the electromagnet isturned on, magnetic particles are collected and the collecting member isthe activated state. When the electromagnetic is turned off, themagnetic particles are released and the collecting member is in thedeactivated state.

“Manipulation”—a collection and release of magnetic particles resultingin their transfer from one location to the other as well as themovements of the particles within a medium for various purposes forexample, for mixing them with various reagents, for rinsing etc.

“Magnetic particles”—particles of various sizes, comprising a magneticsubstance, being a substance which is either a magnet, i.e. having a‘magnetic memory’ or a substance which is not a magnet but is attractedto magnets, i.e. a ferromagnetic material. The magnetic particles mayconsist solely or essentially of the magnetic substance. Alternatively,the magnetic particles may be composite particles comprising themagnetic substance and other non-magnetic substances such as agar,agarose, non-magnetic metal, glass, nitrocellulose, etc. The compositeparticle may either consist of a core or be made of the magneticsubstance and a shell made of the non-magnetic substance or may compriseseveral sub-particles made of the magnetic substance embedded in thenon-magnetic substance. The term “magnetic particles” is to beunderstood as encompassing also the so-called “magnetic beads” or“magnetic microbeads” used in the literature.

SUMMARY OF THE INVENTION

The present invention of the first aspect termed “the system aspect”concerns a system for transfer of material from source vessels to targetvessels such that material from each source vessel is transferred to adesignated target vessel, the system comprising a plurality ofcollecting members permitting simultaneous transfer of material from anumber of source vessels to one or more target vessels, each collectingmember having an activated state for withdrawing and holding materialcontain in a source vessel and a deactivated state in which any materialheld thereby is released, transition from an activated state to adeactivated state of each collecting member is independently controlled.

The source vessels and target vessels may belong to the same array, forexample, the source vessels may be a first line of 12 wells in a 96-wellarrangement and the designated target vessels may be the second line.Alternatively, the source vessels may be all wells in one array (forexample all 96 wells in a 96-well arrangement) and the target vesselsall the wells in another array of vessels.

The system of the present invention, may further comprise a controldevice, such as a computer, and/or a computer controlled robot, whichenables the individual activation and deactivation of each collectingmember of the system.

The selective collection of material from some vessels present in aplurality of vessels, for example, in a 384-well arrangement, may beuseful in various automatic laboratory procedures, (as will be explainedbelow). Selective collection and release of material by some individualcollecting members may be determined by giving x and y axis parametersof the specific collecting member to be activated or deactivated to acomputer/robot.

If desired, the individual collecting members of the system may bedetachably connected to a frame holding them together, so thatindividual collecting members may be detached and either used separatelyor in another system. For example, in sequencing by hybridizationtechniques or combinatiorial chemistry, several devices from the systemmay be detached, fitted to a smaller frame creating a smaller array ofindividual collecting members. This procedure may be repeated again andagain so that each time only those collecting members which collectedparticles with hybridized sequences are collected and rearranged in newand smaller arrays.

The individual activation of each collecting member has an advantage insome laboratory experimental and diagnostic procedures, and in generalallows greater flexibility of the process.

For example, at times a large array of wells containing various reagentsis prepared in advance for carrying out various detection assays, forexample, for detection of infectious agents or genetic diseases in aplurality of samples. Large laboratory centers buy these arrays ofreagents-containing wells in advance. However, at times, for example ina given day, the number of specific samples. to be diagnosed, may besmaller than the number of wells present in the well array. If astate-of-the-art multi-pipetor is used, due to the “all or none” mode ofits activation (all individual pipetors in the device are activated atonce), all the reagent-containing wells which are not used for assayingthe samples, are nevertheless manipulated by collection and transfer,and are in fact contaminated and wasted. Such a procedure which isrepeated many times during the course of a day, in a plurality ofdifferent reagent wells, causes a vast waste of expensive reagents. Byuse of the system of the invention, it is possible to program that onlysome of the wells are used, for example, if only 50 samples are to bediagnosed and the array is of 96 wells, then it is possible to programcollection only from the first 50 wells and the other 46 wells remainintact for future use.

Another use of the system of the present invention, is in combinationalchemistry, for example, for the preparation of short peptides ornucleotide sequences. Today, the most widely used technique forcombinational chemistry is the “mix-and-split” technique, wherein thepool of solid-phase, non-magnetic beads, on which a synthesis shouldoccur, is divided to two or more parts. To each part a differentchemical moiety, for example amino acid, is added, and then the twoparts are repooled, i.e. are combined again together (“mix”), redivided(“split”), and again to each individual pool a different amino acid isadded. These steps are repeated again and again. Since in each step, thedifferent pools of peptides which are being synthesized, are mixed,divided, and to each part a different amino acid is added, by usingseveral elongation steps, a huge variety of different combinations ofpeptides is created. For example if each time the pool is divided into 2parts, then after n steps of mix-and-split, 2^(n) different species ofsynthesized molecules are prepared. However, the problem is that allthese different species of peptides are present in one mixture, and alarge effort in this combinatorial chemistry technique is required inorder to isolate the peptides of interest (Lam et al., Nature, 354:82-84(1991); Jacob et al, Tibtech, 12:19-26 (1994)).

Against this, by use of the system of the invention, it is possible tostart with an array of wells, wherein each well contains a differentchemical moiety serving as a building unit (such as one species of aminoacid, one species of nucleic acid, or chemical moiety) to be added, tothe molecule synthesized by the combinatorial methods. It is a prioriknown which compound is present in each well. A first building unit ofspecies X (amino acid) may be added by using the system of the inventiononly to half the wells in the array. A first building unit of species Ymay be added to the second half of the wells. Then a second buildingunit of species Z may be added to half of the X-containing peptide andto half of the Y-containing peptide, and a second building unit ofspecies T may be added to the other half of the X- containing speciesand the Y- containing species. This results in 4 types of di-peptides:X-Z, X-T, Y-Z, Y-T. Two altered species of a third building unit can beadded again each time to half of the above di-peptides increasing thenumber of different peptides to 2³ (8). In general, the number ofdifferent combinations in A^(p), wherein A is the number of differentamino acids used and P is the length of the peptide. As can be seen, alarge number of different combinations of peptides is created by addingeach time a different amino acid to each half of the elongatingmolecule, so that if each time a new amino acid is added to half of thesamples, after n step 2^(n) different combinations are created. Ifinstead of dividing the species to two parts, the species are divided tofour parts, then after n step 4^(n) different combinations are created.However, by use of the system of the invention, at the end of thesynthesis process it is well known in which well each final combination(i.e. each polypeptide) resides. So, that although the number ofdifferent combinations while utilizing the system of the invention is aslarge as in the mixed-and-split method, there is no need to invest timein isolating each specific species from among the other, since eachspecific polypeptide is present in a separate well.

By one embodiment, termed “the magnetic embodiment”, the material to becollected or released is magnetic particles and the collecting membersare capable of transferring the magnetic particles by magnetic force. Byanother embodiment termed the “non-magnetic embodiment”, the collectingmembers transfer the material by forces other than magnetic force, forexample by vacuum force.

The arrangement and spacing of the individual collecting members in thesystem of the invention should correspond to the arrangement and spacingof the array of vessels (for example wells) from which the materials arecollected and/or to which they are released.

For example in the “non magnetic aspect” the system may provide aplurality of pipetors, each capable of application of vacuum forceindependently. This may be carried out for example by raise of eachpiston of the pipetor independently using miniature, robot-controlservo-mechanism; Alternatively the raise of the piston of the pipetorsmay be carried out using magnetic force or by simultaneous applicationof vacuum force on all pipetors at once but isolating individualpipetors from the vacuum force by activation of independently closingsealing means in some individual pipetors, so that they are isolatedfrom the universally applied vacuum. By such an arrangement material isnot collected by these individual collecting members.

By another aspect termed “the device aspect” the invention concerns anindividual collecting member which is suitable for transfer andmanipulation of magnetic particles. The device may be used separately oras a part of the system of the invention.

Thus the present invention concerns a device for the manipulation ofmagnetic particles, being particles which are attracted by magneticforce, the device comprising:

an elongated member made of a material which is not affected by amagnetic field, having a particle collecting tip and an elongated lumenwith an end, said end of the lumen being at a distal portion of saidelongated member adjacent to said tip; the lumen slidable accommodatinga magnetic field providing member displayable between a first positionin which the magnetic field providing member is in said distal portionwhereby attraction of said particles to said tip occurs, and a secondposition in a proximal portion of said lumen in which said particles arenot attracted to said tip.

The use of magnetic particles in which the magnetic substance isferromagnetic is generally preferred, for example, particles made ofsuperparamagnetic iron oxide. Such particles are capable of respondingwell to relatively weak magnetic fields, but have essentially nomagnetic memory, that is once the magnetic field is removed they do notmaintain magnetic attraction forces.

The elongated member in accordance with the device aspect is made of amaterial which is not affected by a magnetic force, i.e. a non-magneticnon-ferromagnetic material, for example, plastic, glass, varioussynthetic polymers such as polypropylene and the like.

The member has an open-ended or close-ended particle collecting tip,which is the part which comes into contact with the particles and cancollect them by magnetic force. Preferably, said tip is replaceable, oris covered by a replaceable cover, so that after contact with theparticles and the reaction mixture it can be replaced by a clean orsterile tip or cover. This construction enables easy and inexpensivesterilization of the device.

Extending inside the elongated member is an elongated lumen having anopen or a closed end. If the lumen is open its opening should be of asize which does not allow the field provided member to protrudetherefrom. Said end is at a distal part of said member and close to itsparticle collecting tip.

The lumen accommodates a slidable magnetic field providing member, whichmay be completely composed of a magnet; may be made of a magnetic aswell as a ferromagnetic material or made of a material which can becomemagnetized by an electromagnet, but has no magnetic memory. The membercan also comprise an electromagnet with or without a magnetizable core.Such a magnet allows to apply a precise magnetic force for collection ofprecise amounts of magnetic particles for quantitative orsemi-quantitative collection.

The slidable magnetic field providing member may move between twopositions: a first position where the member is at the distal end of thelumen and hence adjacent to said particle collecting tip, and a secondposition wherein the member is outside of the distal end of the lumenand hence distanced from the particle collecting tip.

In said first position (the activated state), since the magnetic fieldproviding member is adjacent to said tip particles are attracted to thetip and in said second position (deactivated state), since the member isdistanced from said tip, particles are not attracted to the tip, orparticles which were previously attracted are released.

The particle collecting tip has a size which is adapted for the specificusage of the device, for example, where the device is used to collectparticles from a well, it should be of the size of a tip of a standardpipetor.

By one option the tip has tapering sides and a truncated end. Atruncated end is preferable since it ensures that the magnetic particlesare not attracted in large clusters to the tip, since such largeclusters are released easily from the tip and thus it is difficult totransfer all the particles. The truncated end also eases quantitative orsemi-quantitative collection of magnetic particles. By another optionthe tip's sides do not taper at all and are parallel right to their endcreating a cylinder shape.

The material of the tip, or of the disposable cover covering the tip,should be of the type and construction as to avoid maintenance of theparticles by adherence, adherence or absorbance thereto, i.e. anon-porous material. As indicated, either the tip is replaceable, or thetip has a cover (similar for example to the plastic tip of an automaticpipetor) which is replaceable. An example of a tip or a replaceablecover are made of polypropylene.

The magnetic field providing member is preferably elongated.

By a preferred embodiment, the body of the field providing member is anelongated magnetic rod, and the end of the rod may be tapered orcylindrical and made of a ferromagnetic material, such as iron ormagnet. The purpose of the iron tapering end is to focus the magneticfield produced by the magnetic rod.

The field providing member may be displaced between the two positionsmanually and for this purpose the field providing member should befitted with a handle.

Alternatively and preferably, the field providing member may bedisplaced automatically, for example, by a computer-controlledmechanical device, or by the aid of a computer-controlled pneumaticpump. The member may also be displaced by the use of an electromagnet,which when turned on, attracts the member to said second position, andwhen tuned off does not attract the member so that it can fall bygravity force, into said first position when the device is held normalto the vessel.

The system of the invention according to the magnetic embodiment maycomprise a plurality of individual devices of the invention. The systemmay comprise any number of devices such as five, eight, ninety-six, etc.(preferably multiples of ninety-six or of 5, 8 or 12), for thesimultaneous manipulation of magnetic particles, for example for thesimultaneous transfer of magnetic particles present in an array ofreaction vessels to another array.

The magnetic field providing members of the individual devicesconstituting the system of the invention may be connected to each otherso that they can be displaced, manually or automatically all together.However, it is preferable that the displacement of each individualdevice constituting the system may be carried out independently so thatit is possible to displace some devices or collecting members in thesystem while not displacing others. Such an arrangement can enable thecollection of magnetic particles from some wells in an array of reactionvessels while magnetic particles from other wells are not collected.Selective collection from specific wells may be useful in variouslaboratory procedures such as sequencing by hybridization (SBH) orcombinatorial chemistry.

By another embodiment of the present invention the magnetic fieldproviding member is stationary inside the elongated member. The magneticfield providing member is either connected to an electromagnet orcomprises an electromagnet. By turning the electromagnet on and off themember becomes magnetized and non-magnetized and magnetic particles areattracted and released from the particle collecting tip, respectively,without any requirement of movement inside the elongated member. Such adevice can also be part of the system of the invention, i.e. the electromagnets of each collecting member are turned on or off independentlyfrom the other electromagnets so that each collecting member isactivated or deactivated individually.

By another aspect, to “the detection aspect” the present inventionconcerns a method for the detection of biological entities carrying afluorescent label, in a sample, the method comprising:

(i) Providing magnetic particles which can specifically bind to saidbiological entities;

(ii) Contacting the magnetic particles with said biological entitiesunder conditions allowing said specific binding;

(iii) Clustering the magnetic particles by magnetic force, therebycausing fluorescent emission to become concentrated in distinct patches;

(iv) Reading said fluorescent emission, reading above control levelindicating the presence of the biological entities in the sample.

The method of the present invention is for detecting any type ofbiological entities which may become any type of molecule, complexes ofmolecules, or cells present in biological tissues. Examples areproteins, peptides, amino acid sequences, nucleic acids sequences,hormones, enzymes, receptors, ligands, polysaccharides and the like, aswell as molecules which are laboratory produced and which are intendedto be similar to biological molecules obtained from natural sources,such as laboratory produced synthesized peptides, oligonucleotidessynthesized by laboratory, for example by PCR methods, antibodies andthe like. The term biological entities also concerns cells, viruses,plasmids, and various cell organels such as mitochondria, denucleos,etc.

The sample may be any type of liquid media containing the biologicalentities which are to be detected. The sample may be obtained from abiological source, or may be the result of a laboratory manipulationsuch as, for example, a result of a PCR amplification of nucleic acidsequences. The biological entities to be separated are of the type whichcarry a fluorescent label. The fluorescent label may be introduced tothe biological entity, which is laboratory produced, during thesynthesis procedure. For example, when synthesizing nucleic acidsequences utilizing PCR, some nucleic types used in the synthesis maybear a fluorescent label. Another example is amino acid sequencessynthesized on a machine, which are synthesized while using somefluorescently labeled amino acid building units.

Alternatively, the biological entities may carry a fluorescent label, byreacting them with other molecules which carry said label. For example,where the biological entity is a protein, it may be reacted with anantibody carrying a fluorescent label. Where the biological entity is areceptor, it may be contacted with a ligand carrying a fluorescent labeland the like.

The particles, according to the method of the invention, are capable ofspecifically binding to the biological entities. The specific binding,is typically carried out by attaching to the magnetic particle onemember of the pair forming group while the other member of the pairforming group is the biological entity to be detected. For example, ifthe biological entity to be detected is a nucleic acid sequence, thenthe magnetic particle should carry the complementary sequence, where thebiological entity is a protein, the magnetic particle should carry aspecific antibody, where the biological entity carries a biotin, orstreptavidin moiety, then the magnetic particle should carry thestreptavidin, or biotin complementary moiety, respectively. Otherexamples of a pair forming group are a receptor and its ligand, anenzyme and its substrate, a lectin and its specific glycoprotein etc.The fact that the magnetic particle bears a molecule which together withthe biological entity forms a pair forming group, ensures specificbinding of the two to each other.

According to the method of the present invention, the magnetic particlesand the sample containing the biological entities are contacted underconditions allowing said specific binding. For example, where the pairforming group is complementary nucleic acid sequences, the conditionsshould be such which allow a specific hybridization, for exampleslightly elevated temperature, in which only complementary sequenceshybridize, while non complementary sequences remain annealed. In thisstep, various rinsing and washing procedures can be carried out in orderto eliminate non specific binding.

The magnetic particles, are now clustered utilizing magnetic force. Thisis typically carried out by placing inside the vessel holding themagnetic particles a magnetic field providing member having a relativelynarrow end. The lines of magnetic field of said end are such which causeclustering of magnetic particles, and if these magnetic particles arebound to the biological entities carrying the fluorescent label, saidclustering causes the fluorescent emission to become concentrated indistinct patches.

By a preferable mode, said clustering is carried by utilizing the deviceof the invention, in its activated state. In this state, the narrow tipof the device, can cause a clustering of the particles in relativelylarge clusters.

The final step of the method is reading that fluorescent emission of thelabel by a suitable instrument For example, the instrument may detectthe change in wavelength from the transmitted to the emitted wave,caused by the fluorescent label. The precise wavelength shift is, ofcourse, dependent on the fluorescent label used.

By use of the method of the present invention, a defused fluorescentsignal, which is caused by the emission of a fluorescent bearingbiological entities, becomes concentrated in distinct patches, thusincreasing the “signal to noise” ratio, and allowing easier detection.

The method of the present invention is particularly useful for a postPCR detection as will be explained herein below.

The invention will now be illustrated with reference to somenon-limiting examples and drawings:

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show a schematic representation of the device of theinvention in a first position (1A) and a second position (1B);

FIG. 2 shows a schematic representation of the system of the inventionaccording to the magnetic embodiment;

FIG. 3A and FIG. 3B shows a schematic representation of the system inaccordance with the non-magnetic embodiment;

FIGS. 4A and 4B show a schematic representation of a device operated bypneumatic force in a first position 4A and a second position 4B;

FIG. 5 and FIG. 6 show flow charts for using magnetic beads for post-PCRdetection and

FIG. 7 shows products of PCR amplification carrying fluorescent labels,concentrated on magnetic particles).

DESCRIPTION OF SPECIFIC EMBODIMENTS

Reference is now made to FIG. 1 which shows a device in accordance withthe invention 70.

The device includes an elongated member 73 made of a non-magneticmaterial, i.e. polypropylene, having attached a particle collecting tip74 with tapering sides and a blunt end. Said tip 74 is covered by adisposable cover 69 so that sterility of the samples may be maintained.Slidable within the elongated member is a magnetic field providingmember 71, which has a portion 72 made of magnetic material, and atapered end 78 made of the same magnet or of a ferromagnetic materialsuch as iron. The purpose of end 78 is to focus the lines of themagnetic field produced by portion 72 to a fine point.

The slidable member 71 can move between two positions: a first positionshown in FIG. 1A in which the end 78 of magnet 72 is adjacent to aparticle collecting tip 74, so that particles 76 are attracted to thetruncated end of said tip 74. It is possible to construct the device sothat a controlled amount of particles are picked while some particlesare maintained The second position (FIG. 1B) the end 78 of magnet 72 ofthe magnetic field providing member, is distanced from tip 74 so thatparticles 76 are released from said end into the reaction vessel 77. Themagnetic field providing member 71 is fitted with a handle 75 for manualdisplacement from the first position (1A) to the second position (1B).

The displacement may also be carried out automatically, for example withthe use of an electromagnet. The automatic displacement may also becomputer controlled.

It is possible to facilitate the release of magnetic particles 76 fromtip 74 by bringing a magnet 79 in close proximity to the end of vessels77. The magnet 79 may be in close proximity with the vessel, butpreferably it should be brought close to the bottom of the vessel onlywhen member 71 is displaced to the second position shown in FIG. 1B. Thesimultaneous movement of the member 71 away from the particles and ofmagnet 79 towards the particles together with vibrator causesresuspension in vessel 77 which may be advantageous for example, whenrinsing the magnetic particles in the liquid, or when mixing them withvarious reagents or when eluting bound molecules into the liquid. Thetransition from the position in FIG. 1A to FIG. 1B may be for thepurpose of transfer of the magnetic particles or may be solely for thepurpose of rinsing/mixing the magnetic particles.

Reference is now made to FIG. 2 which shows the system of the invention80 in accordance with the magnetic embodiment The system is composed ofa plurality of individual devices (collecting members) as thosedisclosed in FIG. 1, in the present case of eight such devices 81. Forother configurations, the number of the devices may vary, and it mayinclude twenty-five devices in a five-by-five format, ninety-six devicesin a standard eight-by-twelve format, etc. Each individual deviceincludes a magnetic field providing member 83, which may be in a loweredposition, thus collecting magnetic particles, or may be in a raisedposition, not collecting magnetic particles, or releasing magneticparticles previously collected.

The spacing of individual devices 81 in the system 80 corresponds to thespacing, and the arrangement of the individual wells, in an array ofwells 82. In the specific case, the array of wells is an array of eightwells, which is a single row in an eight-by-twelve, 96-well format ofstandard micro-titer plates.

For facilitation of release of magnetic particles, when member 81 israised, an array of magnets 84, corresponding to the array of wells, maybe placed beneath of wells 82. Alternatively, a single flat magnet spansthe whole area of the array of wells.

Each individual device 81 is separately connected to control unit 85.Control unit 85 may raise some field providing members 83 in somedevices 81 of the system, while maintaining others in their lowerpositions, thereby enabling collection of magnetic particles only fromsome wells in array 82, while the other magnetic particles remain in thewell. The control may be by separate mechanical mechanism, which arecontrolled by a central computer, but preferably, the control is carriedout by turning on and off individual electromagnets, each connected tomember 83, so that turning on of an electromagnet attracts member 83 andpulls it up, thus releasing magnetic particles (or not attracting them)and turning off a specific electromagnet allows, due to gravity force,member 83 to fall down, thus enabling attraction of the magneticparticles to the particle collecting tip of each device 81.

Frame 86 holds all of individual devices 81 as a single unit. Allsystems 80 may be raised and transferred from one well arrangement 82 toanother well arrangement for depositing of magnetic particles, or to amembrane for formation of an imprint of magnetic particles.

Individual devices 81 may be separated from the system so that they canbe used separately as single devices or alternatively fitted intoanother system by insertion into appropriate places in the frame. Forexample the second, third and fifth device may be detached from frame 86and fitted to a new and smaller frame. Such rearrangement can beadvantageous in sequencing by hybridization (SBH), procedure, wheresmaller and smaller arrays of magnetic particles should be formed forthe determination of a nucleic acid sequence.

FIG. 3A shows another system of the invention 90 in accordance with thenon-magnetic embodiment. The system comprises eight individual pipetors93 capable of collecting small amounts of liquid by magnetic force. Eachpipetor has a handle 94 which when raised creates a vacuum force in tip96 drawing liquid 97 into the tip. When handle 94 is in a loweredposition liquid 97 is not drawn, or previously drawn liquid is releasedfrom tip 96. The spacing and arrangement of individual pipetors 93corresponds to the spacing and arrangement of individual wells in thevessel array 92. The activation (by raising handle 94) and deactivation(by lowering handle 94) of each pipetor 93 is controlled independentlyby control mechanism 95 for example a computer. The handle can beactivated independently by spring mechanism. Alternatively the handleitself may be made of ferromagnetic material and is raised by anelectromagnet, so that although the force created by the pipetor on thematerial to be transferred is vacuum, said vacuum is produced by raisingthe handle with the end of an electromagnet.

FIG. 3B shows essentially the same system as in FIG. 3A, wherein eachidentical component is marked with the same number as in 3A with a prime(′). Individual pipetors 94′ can become deactivated, by raising thewhole pipetor (not the piston) out of vessels 96′ with the aid ofcontrol means 95′.

Another device 100 of the invention is shown in FIGS. 4A and 4B, whilethe device collects magnetic particles by magnetic force, the transferof the collecting member through one state to the other is carried outby vacuum pressure, i.e. is activated by pneumatic forces. Device 100consists of a magnet 109, which is slidable within the inner lumen ofdevice 100. In FIG. 4A magnet 109 is in contact with the inner wall of aflat polypropylene tip end 110. When the magnet is in contact with thistip (FIG. 4A), magnetic particles 111 are collected by magnetic force tothe tip. The magnet is held by magnetic force from an iron made ring108. When vacuum force is applied, air moves in through opening 106,forcing piston 105 to move up. The piston 105 is connected to magnet 109through connector 107. Thus, when vacuum force is applied (FIG. 4B), themagnet moves from its lower position shown (FIG. 4A) to its upperposition. The magnet remains in the upper position (FIG. 4B) due tomagnetic force applied by upper iron ring 104. In such a position asshown in FIG. 4B, magnetic particles 111 are not collected or arereleased back to the vessel. When air pressure is released from thepistons through upper inlet 103 magnet moves again into the positionshown in FIG. 4A and particles are recollected. 101 is an O-ring usedfor sealing the upper inlet when vacuum force is applied. 102 is anupper closure of device 100.

Optionally, an ultrasonic vibrator can be attached to a 96-wellmicroplate, and this permits resuspension of magnetic particles in thewell.

EXAMPLE 1 Collection and Transfer of Magnetic Particles with the Deviceof the Invention

A. Materials and Tools

The magnetic particles′ suspensions used was 1 mg/ml and 2 mg/ml(BioBeads Merck). The magnetic plate for 96-well microplates wasmanufactured by PerSeptive Bio-System and used with a 96-wellpolypropylene microplate. The magnetic device of the invention(Bio-Magnetics) had a cylindric magnet 4 mm diameter and length 20 mm(NdFeB—N40/Ni from Magma). The polypropylene tips used as disposabletips had flat ends of different thickness 30, 50 and 90μ (micrometer).The microscope slide used for counting of particles was Bright-lineHemacytometer. Microscope was B×60, Olympus. The buffer used was 1×SSC.

B. Experimental Procedures

Three wells in the polypropylene plate were loaded with 0.1, 0.15 and0.2 ml buffer and three adjacent wells were loaded with 0.1, 0.15 and0.2 ml of 1 mg/ml magnetic particles suspension (stock suspension). Thepolypropylene tip with flat end of 90 micrometer was attached on endside of the magnetic device of the invention.

The device was held over a well No. 1 containing the suspension (0.1 ml)(the magnet contacts the inner side of the 90 micrometer flat tip) andmoved so it gently touched the suspension surface for one minute.Magnetic particles moved towards the tip and created a “button” composedof a cluster of magnetic particles. As a result the suspension becameclear. Next, the device is taken out of the well, and the microplate isput on the flat magnet in order to facilitate detachment of theparticles from the device re-suspension of the collected magneticparticles to the wells.

In order to move the cylindric magnet to the second position (where themagnet is distanced from the particle collecting end) by magnet force aniron rod was placed inside the device.

Then the device was brought over a well with 0.1 ml buffer so that theflat tip was gently touching the buffer. Particles moved down into thebuffer with the aid of magnetic field from the flat magnet in less thana minute.

The remaining particles in well No. 1 were mixed with a pipette and a0.05 ml sample was taken from the homogeneous solution and put on a cellcounting glass. the results are shown in the following Table 1:

TABLE 1 m.p. m.p. Efficiency Thickness Stock Stock Buffer remaining instock of device No. of tip concentration volume volume in 10⁻⁴ml in 10⁻⁴ml % 1 30μ 1 mg/ml 100 μl 100 μl 0.2 1000 99.98 150 μl 150 μl 1 99.90200 μl 200 μl 0.8 99.92 2 50μ 1 mg/ml 100 μl 100 μl 0.8 1000 99.92 150μl 150 μl 3 99.70 200 μl 200 μl 5.4 99.46 3 90μ 1 mg/ml 100 μl 100 μl16.7 1000 99.33 150 μl 150 μl 5 99.50 200 μl 200 μl 7.2 99.28 4 30μ 2mg/ml 100 μl 100 μl 0.4 2000 99.98 150 μl 150 μl 3.33 99.83 200 μl 200μl 3 99.85 5 50μ 2 mg/ml 100 μl 100 μl 1.8 2000 99.91 150 μl 150 μl 3.499.83 200 μl 200 μl 0.4 99.98 6 90μ 2 mg/ml 100 μl 100 μl 5.33 200099.73 150 μl 150 μl 12.4 99.38 200 μl 200 μl 6.4 99.68

For determination of magnetic particles counting under the microscopetook place. The efficiency of the magnetic device is calculatedaccording to the formula: % yield=100%* (remaining m.p. in 50 μl aftercollection)/(m.p. in 50 μl stock suspension)

Efficiency of device=100%—% yield (efficiency means % of transferredm.p.).

The above experiment was repeated under the following conditions:

with 0.15 and 0.2 ml suspension

with 30 and 50 micrometer thick tip and

with 2 mg/ml particle suspension

EXAMPLE 2 Use of the System of the Invention for Creation of a PeptideLibrary by Combinatorial Chemistry Materials

1. Four polypropylene 96-well microtiter plates (Nunc, Denmark)containing four different amino acids depicted schematically as (A, B,C, D) blocked at the amino end by tertbutyloxycarbonyl (Boc) Group,(each plate contains one amino acid in approximately 16 pmol per well)together with dicyclohexylcarbodiimide in a total volume of 200 μl perwell.

2. 96-well microtiter plate with magnetic particles suspensions, 200 μlper well (200 μg particles). The particles are used as solid phase forpeptide synthesis based on the Merrifield method.

3. 96-well microtiter plate with 50 per cent trifluoroacetic acid indichloromethane, 200 μl per well.

4. 96-well microtiter plate with diisopropyl ethyl amine, 200 μl perwell.

5. 96-well plate with 200 μl HF per well.

6. A system of 96 magnetic devices (or pins) corresponding to 96-wellformat. This is Bio-Magnetics unique combinatorial system (orcombinatorial pin device).

Experimental Procedures

Step 1

1. The first amino acid (20 pmol/mg magnetic particles) is already boundto the magnetic particles via their carboxyl end and the amino group ismasked (or blocked) with a protecting group. The system of 96 pins isused to collect simultaneously magnetic particles from 64 wells andtransfer them to a plate with 50% trifluoroacetic acid indichloromethane. The Boc Group is completely removed, with minimal lossof the other protecting groups.

2. The system of 96 pins is used to collect particles from all the 64wells and transfer them to a plate with diisopropyl ethyl amine. Thistertiary amine neutralizes the α-amine salt (created in step 1) and thefree amine of the bound amino acid is then ready to couple with a secondBoc-amino acid.

As a result is created 64 wells all containing the same amino acid.

64 wells

Step 2

3. The system of 96 pins is used to collect particles from 16 wells andtransfer them to 16 wells in a plate containing amino acid A. The secondgroup of particles are transferred from 16 wells to 16 wells in a platecontaining amino acid B. The third group of particles are transferred to16 wells in a plate containing amino acid C. The fourth group ofparticles are transferred to 16 wells in a plate containing amino acidD. In each of the four plates the dicyclohexylcarbodiimide activates thecoupling reaction. The result is four groups of different dipeptides,each group containing 16 dipeptides. As a result 4 different groupsconsisting of 16 wells are created.

A B 16 wells C D

4. Magnetic particles from all 64 wells (the four groups) are collectedvia the 96 pin system (device) and transferred to a plate containingtrifuoroacetic acid in dichloromethane.

5. Magnetic particles from 64 wells are collected with the 96 pin systemand transferred to 64 wells in a plate containing diisopropyl ethylamine.

Step 3

6. Each group of 16 wells is divided to four subgroups (each subgroup offour wells). Using the 96 pin system magnetic particles from eachsubgroup are transferred to a plate with different amino acid. Onesubgroup to amino acid A, the second subgroup to amino acid B, the thirdsubgroup to amino acid C, the fourth subgroup to amino acid D. In factthe 96 pin system collects simultaneously four subgroups, each onecorresponding to a different group of 16 wells (See No. 3). The resultis 16 different tripeptides in each group of four wells.

A B A B 4 wells D C D A B A B C D C D

Step 4

7. Steps 4 and 5 are repeated.

8. Each subgroup contains four wells with the same tripeptides. The 96pin system collects particles simultaneously from 16 wells (each wellfrom a different subgroup) and transfers them to a plate containingamino acid A. Particles from 16 other (each well from a differentsubgroup) wells are collected via the 96 pin system and transferred to aplate containing amino acid B. The same action is done with the rest 32wells to transfer them to plates with amino acid C and D. The resultsare 64 different tetrapeptides bound to magnetic particles.

A B A B C D C D well

9. If needed the peptides can be separated from the particles. This canbe done by transferring the particles to a plate containing a stronganhydrous acid HF. Then the particles can be collected leaving thepeptides in solution.

To Summarize

In the Mix-and-split method the resulting peptide libraries are mixedlibraries. The new method of Example 3 permits creating orderedlibraries where the location of each peptide is known. An orderedlibrary can save work and time when detecting unknown peptide in a mixedlibrary.

EXAMPLE 3 Post PCR Detection

The schematic flow charts for carrying out the detection method of theinvention are shown in FIGS. 5 and 6.

A. Materials

I Template

PCR Fragment of 152 bp of the Exon 6 of Human amiloride-sensitiveepithelial sodium channel beta subunit gene.

II Oligonucleotides:

a. E1: biotin-10dT-aagcaacccctctaaacacag (SEQ ID NO:1), which was usedas forward primer in amplification of the template;

b. E2: biotin-10dT-aggcgtgcaccaccttcccac (SEQ ID NO:2), which was usedas reverse primer in amplification of the template;

c. C1: biotin-10dT-cctgaaccgctctatacacag (SEQ ID NO:3), a control to E1

d. C2: A control of BioBeads Streptavidin, without boundoligonucleotides.

All the oligonucleotides were synthesized in BTG Israel.

III Magnetic particles:

BioBeads Streptavidin from Merck, 10 mg/ml with a binding capacity forbiotinylated oligonucleotide of 400 pmol/1 mg full capacity or 20pmol/50 μg.

B. Experimental Procedures

IV Coupling of oligonucleotides to BioBeads:

300 μg/ml oligonucleotides were dissolved in 20 mM Tris-HCl buffer pH8.0 and were diluted in binding buffer (Dynal 2M NaCl, 1 Mm EDTA, 10 mMTris-HCl pH 7.5) and added to 100 μg magnetic particles (m.p.). Thecoupling reaction was carried out at 37° C. for 30 min. with continuousshaking. 50 μg of magnetic particles were coupled with 0.1 pmole ofoligonucleotides.

V Fluorescent Labeling of PCR product/template:

AMCA-6-dUTP (Amino-methylcoumarin-6-2′-deoxyuridine-5′-triphosphate)from Boehringer Mannheim was used to label the template DNA in a PCR.The PCR product was further analyzed by PAGE to verify amplification.1-20 μl of PCR product were used in different hybridization reactions.

VI Hybridization on magnetic particles (M.P.)

30 μg of magnetic particles were prehybridized in 8 μl of hybridizationbuffer at 45° C. for 20′, Hybridization buffer contained: 5×SSC pH 7,Sambrook, Fritsch & Maniatis MOLECULAR CLONING: A LABORATORY MANUAL 19892/E and 0.25 SDS. Hybridization of 2 μl fluorescent PCR product to theimmobilized oligonucleotides on the m.p. was carried out at 60° C. for5′, 15′, 45′ and 60′. Following hybridization the supernatants weredecanted, and the particles were washed with wash buffers: once with5×SSC buffer containing 0.2% SDS at 55° C., twice with 1×SSC buffercontaining 0.2% SDS at 55° C. and once with 1×SSC buffer at 65° C. Atthe end of the experiment the m.p. were resuspended in 1×SSC buffer at aconcentration of 1 μg/μl and 1.0 μl was then analyzed under fluorescentmicroscope.

C. Results

(i) Fluorescence Microscopic Observations:

Surface fluorescence on the magnetic particles was assessed using B×60Olympus Microscope with 100 W Hg lamp 330-385 mm Excitation filter, DM400 Dichroic mirror, BA 420 barrier filter.

(ii) Effect of Temperature on Hybridization

Hybridization at lower temperature of 42° C. and 55° C. with subsequentwashing at 4° C. did not give adequate specific binding. Hybridizationat 60° C. was successful in getting fluorescent positive signal. Washingthe particles at higher temperature than that of hybridization itselffurther substantially reduced the background of non-specific binding.

(iii) Effect of Concentration of Immobilized Oligonucleotide on m.p.

Our observations suggested that when magnetic particles were coupled tothe oligonucleotides at 100% binding capacity (20 pmol/50 μg m.p.) or50% binding capacity (10 pmol/50 μg m.p.); the hybridization reactionwas not successful. We observe absolute no fluorescently labeledparticles or undetectable fluorescence on the particles. We suggest thiscould be due to the steric hindrance created by the boundoligonucleotides on the particles. When the amount of boundoligonucleotides was reduced to 1 pmol/50 μg m.p. and 0.1 pmol/50 μgm.p. a significant increase in number of fluorescently labeled magneticparticles was observed. This effect was although more obvious with the0.1 pmol/50 μg m.p.

(iv) Effect of the Poly(T) Spacer on Hybridization

Oligonucleotides bound without spacer (linker arm) gave negativeresults. However, use of 10(dT) linker gave adequate binding to thetemplate DNA.

(v) Effect of Magnetic Concentration

The presence of SDS during hybridization (0.25 % SDS) and subsequentwashing (0.2% SDS) improved specific binding conditions.

(vi) Effect of Magnetic Concentration

Positive fluorescent signals, mean that part of the magnetic particlesare fluorescent and the others are black/brown. Sometimes if the numberof fluorescent particles is low compared to the black particles it ispreferable to magnetically concentrate the particles. The aggregate ofparticles permits getting a stronger fluorescent signal (compared todispersed particles). Following are CCD Camera photographs of magneticparticles aggregates. In FIG. 7 the upper photograph is blue fluorescentemission from thousands of particles after specific hybridization of 60′in 60° C. Control 1 (Con 1) and Control 2 (Con 2) gave no emission,because non specific binding did not occur. In page 2 the same sampleswere analyzed with visible transmitted light to get pictures of theaggregates.

References:

1. Immobilization of polynucleotides on magnetic particles, Philip J. R.Day, Pargat S. Flora, John E. Fox and Matthew R. Walker, Biochem. J.278:735-740, (1991).

What is claimed is:
 1. A system for transfer of solid magnetic particlesfrom a plurality of source vessels to a plurality of target vessels suchthat material from each source vessel is transferred to a designatedtarget vessel, the system comprising a plurality of detachably connectedcollecting members permitting simultaneous transfer of solid particlesfrom a number of source vessels to one or more target vessels; eachcollecting member is made of a material which is not affected by amagnetic field, having a truncated particle collecting tip and anelongated lumen with an open end, said end of the lumen being at adistal portion of said elongated member adjacent to said tip; the lumenaccommodating a magnetic field providing member within the lumen, whichmember is displaceable between a first position in which the magneticfield providing member is in said distal portion whereby attraction ofsaid particles to said truncated tip occurs, and a second position in aproximal portion of said lumen in which said particles are not attractedto said truncated tip, transition between the first position and thesecond position is controlled by electric or pneumatic force; eachcollecting member is independently controlled.
 2. A system according toclaim 1, further comprising a computer for controlling the independenttransition from said first position to said second position of eachcollecting member.
 3. A system according to claim 1, wherein the firstposition is obtained by application of a force capable of creating amagnetic field in the truncated tip of each collecting member and thesecond position is achieved by ceasing of creation of said magneticfield.
 4. A system according to claim 3, wherein the magnetic field iscreated by the use of an electromagnet present in each collectingmember.
 5. A system according to claim 3, wherein the magnetic field iscreated by the use of a pneumatic pump.
 6. A system according to claim3, wherein the magnetic field is created by the use of a spring.
 7. Asystem according to claim 1 for the quantitative collection of magneticparticles attracted to the truncated tip, wherein the proportion ofparticles collected is in correlation to the time of collection.
 8. Asystem for quantitative collection of magnetic particles according toclaim 7, wherein the truncated tip thickness is less than 30 μm.